(without serum) mixed with 3 μg plasmid diluted in 100 μL
DMEM (without serum) for each well to transfect. The transfection mixes (200 μL per well) are incubated for 5–15 min at
room temperature (RT) and then added drop wise to their
corresponding wells. The cells are then incubated overnight
at 37
C, 5% CO 2 .
4. On the next day, remove the medium from each well and
replace by 2 mL of DMEM containing 50 μM biotin and
200 ng mL
À1 doxycycline if using inducible plasmids under
the control of a tet-responsive element (see Note 16). Incubate
the cells for at least 16 h at 37
C, 5% CO 2 .
5. On the following day, wash cells in each well with 1 mL ice-cold
PBS and then scrap the cells in 100 μL of lysis buffer I with
disposable cell scrappers.
6. Transfer each sample of scrapped cells to 1.5 mL tubes, resuspend briefly by pipetting and centrifuge at 14,000 Â g for
10 min at 4
C to clear the lysates.
7. Transfer the supernatants to fresh 1.5-mL tubes and place
on ice.
8. Determine the protein concentration of each sample using a
standard Bradford assay.
9. Prepare samples for SDS-PAGE by mixing an equal amount
(15–30 μg protein content) of each lysate with 5Â SDS-loading
buffer to yield a total of 30 μL SDS-PAGE sample in 1Â
SDS-loading buffer (volume are adjusted with lysis buffer I).
Denature the samples at 95
C for 5 min (see Note 17).
10. Load the samples (20 μL/lane) and molecular weight markers
on an SDS-polyacrylamide gel with a percentage allowing the
appropriate resolution of the different fusion proteins.
11. After electrophoresis, transfer the fractionated proteins to an
immunoblotting membrane (see Note 9).
12. Following protein transfer, block the membrane with 5% dry
milk in PBS for 30 min at RT.
13. To detect biotinylated proteins, incubate the membrane in a
50-mL falcon tube with 5 mL of conjugated streptavidin
diluted 1:15,000 in PBS containing 2% bovine serum albumin
(BSA) and 0.1% Tween-20 for 30 min at RT on a roller-shaker.
14. Wash the membrane three times with PBS containing 0.1%
Tween-20 for 10 min at RT (see Note 18) and then proceed
with detection of chemoluminescence or fluorescence signals
(Fig. 3).
15. If detection of the fusion proteins is desired, incubate the
membrane in a 50-mL falcon tube with 5 mL of anti-Myc
(to detect the fusion to NBirA*) and anti-FLAG (to detect
Context-Specific Proximity-Dependent Proteomics with Split-BioID
309
DMEM (without serum) for each well to transfect. The transfection mixes (200 μL per well) are incubated for 5–15 min at
room temperature (RT) and then added drop wise to their
corresponding wells. The cells are then incubated overnight
at 37
C, 5% CO 2 .
4. On the next day, remove the medium from each well and
replace by 2 mL of DMEM containing 50 μM biotin and
200 ng mL
À1 doxycycline if using inducible plasmids under
the control of a tet-responsive element (see Note 16). Incubate
the cells for at least 16 h at 37
C, 5% CO 2 .
5. On the following day, wash cells in each well with 1 mL ice-cold
PBS and then scrap the cells in 100 μL of lysis buffer I with
disposable cell scrappers.
6. Transfer each sample of scrapped cells to 1.5 mL tubes, resuspend briefly by pipetting and centrifuge at 14,000 Â g for
10 min at 4
C to clear the lysates.
7. Transfer the supernatants to fresh 1.5-mL tubes and place
on ice.
8. Determine the protein concentration of each sample using a
standard Bradford assay.
9. Prepare samples for SDS-PAGE by mixing an equal amount
(15–30 μg protein content) of each lysate with 5Â SDS-loading
buffer to yield a total of 30 μL SDS-PAGE sample in 1Â
SDS-loading buffer (volume are adjusted with lysis buffer I).
Denature the samples at 95
C for 5 min (see Note 17).
10. Load the samples (20 μL/lane) and molecular weight markers
on an SDS-polyacrylamide gel with a percentage allowing the
appropriate resolution of the different fusion proteins.
11. After electrophoresis, transfer the fractionated proteins to an
immunoblotting membrane (see Note 9).
12. Following protein transfer, block the membrane with 5% dry
milk in PBS for 30 min at RT.
13. To detect biotinylated proteins, incubate the membrane in a
50-mL falcon tube with 5 mL of conjugated streptavidin
diluted 1:15,000 in PBS containing 2% bovine serum albumin
(BSA) and 0.1% Tween-20 for 30 min at RT on a roller-shaker.
14. Wash the membrane three times with PBS containing 0.1%
Tween-20 for 10 min at RT (see Note 18) and then proceed
with detection of chemoluminescence or fluorescence signals
(Fig. 3).
15. If detection of the fusion proteins is desired, incubate the
membrane in a 50-mL falcon tube with 5 mL of anti-Myc
(to detect the fusion to NBirA*) and anti-FLAG (to detect
Context-Specific Proximity-Dependent Proteomics with Split-BioID
309